Cholangiocarcinoma (CCA)-associated fibroblasts (CCFs) have been identified the cancer progression impact through the aberrant production of tumorigenic substances. This study is aimed to investigate the production of tumor-related cytokines from primary culture CCFs and to characterize tumorigenic induction properties. Primary culture CCFs were isolated from fresh CCA tissues and verified by morphology, chromosomal pattern, and the expressions of fibroblast markers: vimentin (VIM), alpha-smooth muscle actin (ASMA); epithelial markers: cytokeratin 7 and 19 (CK7 and CK19); and periostin (PN). Migration induction of the conditioned-media (CM) from CCFs on CCA cells was shown by wound healing assay. The results revealed that all CCFs had the spindle-liked morphologies and the presence of VIM, ASMA and PN, but no CK19. CCFs-CM induced migration of CCA cells. CCFs had aberrant chromosomal pattern including tetraploid and diploid chromosomes with deletion, amplification and translocation. The abnormal karyotypes were 92,XXYY; 46,X,-Y,+7; 46,XY,der(8)t(8;?); 92,XXYY,der(8)t(8;?),der(8)t(8;?); and 92,XX,-Y,-Y,+7,+7. In contrast, normal skin fibroblasts (NLFs) exhibited diploid chromosome with 50% had Y-chromosome deletion. Interleukin-6 (IL-6) and hepatocyte growth factor (HGF) located on chromosome 7 showed increased expressions and IL-6 was confirmed the increment in more CCF-CMs. These observations provide the evidences that dysregulation of IL-6 which is a tumorigenic substances from CCFs may be the result from chromosomal aberrations. These data give us an experimental foundation for further studies to explore the mechanism underlined the involvement of fibroblasts in CCA progression.